肠道病毒A组71型病毒参考品的建立及应用

Establishment and application of reference material for enterovirus A71

目的建立肠道病毒A组71型(enterovirus A71,EV-A71)灭活疫苗病毒参考品,用于病毒滴度内部质控品及灭活效果验证实验室评价。方法建立1批病毒参考品,按照《中华人民共和国药典》对其进行检定,包括无菌、支原体及分子生物学鉴定检查等。对其滴度进行标定和储存稳定性研究,并进行病毒滴定和灭活效果验证方面的应用研究。结果建立的EV-A71病毒参考品无菌检查及支原体检查结果均符合规定。分子生物学鉴定结果显示参考品只含有单一的EV-A71 VP1特异性条带。EV-A71病毒参考品标定后的滴度平均值为7.000 lgCCID_(50)/ml(95%CI为6.917~7.083 lgCCID_(50)/ml),可接受范围为6.566~7.434 lgCCID_(50)/ml,变异系数(coefficient of variation,CV)为3.01%,于-60℃及以下存放12个月的CV为2.08%,稳定性良好。将参考品作为病毒滴定测定的内部质控品应用于日常监测50次试验,滴度趋势分析结果显示未出现超趋势现象(out of trend,OOT)且均在警戒限内;将参考品作为阳性对照应用于灭活效果验证,结果显示细胞感染法及免疫荧光法的检测结果较为一致。结论建立了EV-A71病毒感染性滴度检测用参考品(EV-A71病毒参考品),12个月内储存稳定性良好,可用于EV-A71灭活疫苗生产工艺中病毒滴度评价和控制,以及作为灭活效果验证中的阳性对照,从而为单价或多价肠道病毒灭活疫苗的研究提供理论依据。

Objective To establish the enterovirus A71(EV-A71)reference material for internal quality control of virus titration and validation of inactivation effect.Methods A batch of virus reference material of EV-A71 was established.According to the Chinese Pharmacopoeia,sterility,mycoplasma,and molecular biological identification were carried out.Three experimenters collaboratively calibrated EV-A71 reference material,and its stability was evaluated for 12 months.The application of virus titration and inactivation effect verification was also evaluated.Results The detecting results of sterility and mycoplasma tests of the reference material were all accorded with standards.Molecular biological identification revealed only a single,specific band of EV-A71 VP1.The average titer of the working virus seed bank was 7.000 lgCCID_(50)/ml(95%CI:6.917—7.083 lgCCID_(50)/ml).The acceptable range of external quality was 6.566—7.434 lgCCID_(50)/ml,and the coefficient of variation(CV)was 3.01%,EV-A71 reference showed good stability stored under -60℃ for 12 months,with a CV of 2.08%.The viral titer internal control was used for daily monitoring of 50 tests;the trend analysis of virus titers showed that no outof-trend(OOT)was observed and all were within the warning limits.It was used as a positive control to the verification of virus inactivation;the immunofluorescence results were consistent with the results of the cell infection method.Conclusions The EV-A71 virus reference material is established,which has good storage stability within 12 months.It can be used to evaluate and control viral titer in the production process of EV-A71 inactivated vaccine and as a positive control in the validation of virus inactivation effects.It provides a foundation for developing monovalent or polyvalent inactivated vaccines against enterovirus.